The long term objective of my laboratory is to understand the mechanism(s) of insulin resistance in non-insulin dependent diabetes mellitus (NIDDM). Although the precise mechanism of insulin resistance in NIDDM has not been fully elucidated, studies from various laboratories, including ours, suggest abnormalities in insulin binding, transmembrane signalling and glucose transport effector system. Some of these abnormalities are undoubtedly acquired but the genetic abnormalities in the two most obvious candidate genes (insulin receptor and glucose transporter) cannot be ruled out as an etiology in certain cases of NIDDM. Mutations in the insulin receptor gene have been identified in patients with various genetic syndromes and extreme insulin resistance. Transfection studies with these mutant insulin receptor cDNAs demonstrate various functional abnormalities. The specific aims for this pilot project are to (i) investigate the possible mutation(s) in the insulin receptor gene from NIDDM patients with a strong family history of NIDDM, and then to (ii) determine the biological significance of such mutation(s) in the structure and function of insulin receptor by transfection of receptor cDNAs into NIH 3T3-Ll cells. The mutation(s) in the insulin receptor gene will be determined by direct nucleotide sequencing of either genomic DNA or cDNA of the insulin receptor isolated from affected and non-affected family members using polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) methodologies. During the course of training, the PI will have hands on experience in (i) preparations of mRNA and genomic DNA, (ii) PCR amplification of mRNA and genomic DNA, (iii) direct sequencing of DNA, (iv) Southern blot analysis, (v) Northern blot analysis and Sl nuclease protection assay, (vi) allele-specific oligonucleotide hybridization, and (vii) transfaction of mutant insulin receptor cDNA. Such a comprehensive introduction to molecular biology and recombinant DNA technology in Dr. Simeon Taylor's laboratory will enable the PI to gain experience in the area of molecular biology and to study mutations in the glucose transporter(s) gene or other possible candidate genes in patients with NIDDM.